Edema is the term generally used to describe the accumulation of excess fluid in the intercellular (interstitial) tissue spaces or body cavities. Edema may occur as a localized phenomenon such as the swelling of a leg when the venous outflow is obstructed; or it may be systemic as in congestive heart failure or renal failure. When edema is severe and generalized, there is diffuse swelling of all tissues and organs in the body and particularly pronounced areas are given their own individual names. For example, collection of edema in the peritoneal cavity is known as "ascites"; accumulations of fluid in the pleural cavity are termed "hydrothorax"; and edema of the pericardial sac is termed "pericardial effusion" or "hydropericardium". Non-inflammatory edema fluid such as accumulates in heart failure and renal disease is protein poor and referred to as a "transudate". In contrast, inflammatory edema related to increased endothelial permeability is protein rich and is caused by the escape of plasma proteins (principally albumin) and polymorphonuclear leukocytes (hereinafter "PMNs") to form an exudate.
Edema, whether inflammatory or non-inflammatory in nature, is thus an abnormality in the fluid balance within the microcirculation which includes the small arterioles, capillaries, and post-capillary venules of the circulatory system. Normal fluid balance and exchange is critically dependent on the presence of an intact and metabolically active endothelium within the vasculature. Normal endothelium is a thin, squamous epitbelium adapted to permit free, rapid exchange of water and small molecules between plasma and interstitium; but one which limits the passage of plasma proteins with increases in protein size.
The endothelial lining of all arterioles and venules, and most capillaries in the body, is of the continuous type, having an unbroken cytoplasmic layer with closely apposed intercellular junctions. Physiological studies [Renkin, E., Circ. Res. 41:735-743 (1977); Renkin, E., ACTA Physiol. Scand. (Suppl.) 463:81 (1979); Bottaro et al., Microvasc. Res. 32:389-398 (1986)] have demonstrated normal endothelial permeability for water and small molecules by the existence of water-filled small pores approximately 6 nanometers (hereinafter "nm") in radius or by slits about 8 nm wide. There is also believed to be a system of larger sized pores about 25 nm in radius which accounts for the small quantities of protein and other large solutes that normally cross the endothelial wall barrier.
A variety of different disturbances can induce a condition of edema. These include: an elevated venous hydrostatic pressure which may be caused by thrombosis of a vein or any other venous obstruction; hypoproteinemia with reduced plasma oncotic pressure resulting from either inadequate synthesis or increased loss of albumin; increased osmotic pressure of the interstitial fluid due to abnormal accumulation of sodium in the body because renal excretion of sodium cannot keep pace with the intake; failure of the lymphatics to remove fluid and protein adequately from the interstitial space; an increased capillary permeabiity to fluids and proteins as occurs in the inflammatory response to tissue injury; an increased mucopolysaccharide content within the interstitial spaces; and an iatrogennic induced or mediated increase or accumulation of fluid and protein resulting from the administration of a pharmacologically active lymphokine during the course of treatment by a physician or surgeon. This last-identified disturbance merits a more detailed examination in view of recent developments in immunotherapy.
Lymphokines comprise a broad class of biologically active substances which are secreted by various types of lymphocytes in-vivo and in-vitro, especially by different populations of T-cell lymphocytes. Perhaps the most controversial presently is the family of interleukins, which presently comprises: Interleukin-1 ("IL-1") involved in the activation of resting T-cells; Interleukin-2 ("IL-2") which mediates the proliferation of T-cells and induces cytotoxic activity in T-cells; Interleukin-3 ("IL-3") which causes the proliferation of mast cells and granulocytes; Interleukin-4 ("IL-4") which mediates the proliferation, activation, and differentiation of B-cells, T-cells, and natural killer cells; and Interleukin-6 ("IL-6") which induces the growth and differentiation of B-cells and T-cells [Spits et al., J. Immunol. 139:1142 (1987); Kawakami et al., J. Exp. Med. 168:2183 (1988); Spits et al., J. Immunol. 141:29 (1988); and Tartakovsky et al., J. Immunol. 141:3863 (1983)].
Of particular interest is Interleukin-2 ("IL-2") and its analogues whose isolation, chemical formulation and structure, and synthesis by conventional wet chemistries and by recombinant DNA techniques have been intensively pursued [see for example U.S. Pat. Nos. 4,490,289; 4,138,927; 4,569,790; 4,578,335; 4,761,375; 4,518,584; 4,604,377; 4,564,593; and the references cited therein]. The use of Interleukin-2 as a therapeutic composition has followed two very different approaches. The first line of development is exemplified by the IL-2 immunotherapy technique for treating cancers originated by Dr. Steven A. Rosenberg [Rosenberg, S. A., Immuno. Today 9:58-62 (1988) and the references cited therein]. That approach involves the removal of lymphoid cells from a tumor-bearing host; an in-vitro expansion of the host's cells by culture in Interleukin-2 containing culture media to produce lymphokine-activated killer ("LAK") cells; and a re-introduction of the LAK cell culture into the living host accompanied by successive administrations of IL-2 directly to the tumor-bearing host. This approach has been termed "adoptive immunotherapy." A variation of this development is the use of IL- 2 directly as an anti-cancer agent. This is exemplified by Patent Nos. WO-8600334 published 860116; JP-60185721 published 850921; U.S. Pat. No. 4,645,830; EP-145390 published 850619; and WO8500606 published 850214.
The second general approach has been to chemically link the Interleukin-2 molecule to another ligand having known biological properties to form a hybrid. The IL-2 portion of the hybrid serves a specific binding protein able to selectively bind to IL-2 receptor sites on the surface of living cells; the IL-2 portion of the hybrid thus served as the means of delivering and selectively introducing the other component of the hybrid into a chosen cell population having IL-2 receptor sites on their cell surface. This second approach is exemplified by: U.S. Pat. Nos. 4,675,382 and 4,745,180; GB-2189393 published 871028; EP-269455 published 88060; EP-256714 published 880224; and EP236987 published 870916.
A major problem and deficiency for the in-vivo use of interleukins generally and of IL-2 in particular has been the now well recognized phenomenon of uncontrolled edema as a concomitant side-effect of interleukin administration. The observation of massive edema locally and systemically has been termed "vacular leakage syndrome" and is now seen as a regular and unavoidable consequence of using interleukins therapeutically in either uncoupled or complexed/hybridized form. Representative publications describing the uncontrolled edemas caused or mediated by the administration of an interleukin include: Carlsen, E. and H. Prydz, Thrombosis and Harmostasis 58:257 (1987); Giddings, J. C. and L. Shall, Thrombosis and Haemostasis 58:31 (1987); Kotasek et al., Clin. Res. 35:660A (1987); Ellison et al., Anat. Rec. 218:41A (1987); and the references cited therein]. In so far as is presently known, there is no effective composition or method to meaningfully control, reduce, or substantially eliminate the resulting vascular leakages and the massive edemas caused and/or mediated by the in-vivo administration of an interleukin for any therapeutic purpose. Furthermore, there is presently no effective agent or admixture of substances useful as a prophylactic against an interleukin-mediated edema.
Remote from and completely unrelated to these investigations of interleukins were other research efforts directed towards the isolation and identification of the component substances of the poisonous green fungus Amanita phalloides, known as the "green death cap" or "deadly agaric" mushroom [Lynen and Wieland, Justus Liebigs. Ann. Chem. 533:93-117 (1938); Wieland and Schnabel, Justus Liebigs Ann. Chem. 657: (1962)]. At least ten peptide-like substances of complex structure have been identified; most of these substances have proven to be extremely toxic liver toxins [Liebig's Ann. Chem., volume 617, page 152, 1958; Pharmacol. Reviews, volume 7, page 87, 1959; Liebig's Ann. Chem., volume 704, page 226, 1967]. Upon isolation and empirical analysis of the naturally occurring individual components of Amanita phalloides, however, investigators found at least two different naturally occurring classes of chemical compositions: phallotoxins and antamanide.
The class of phallotoxins as a whole is best exemplified by the substance known as phalloidin, one of seven naturally occurring member toxins forming the class. Phalloidin is very rapid in action. High dose levels given intramuscularly cause death of mice or rats within one or two hours. The LD.sub.50 dose in albino mice is only 3.3 micrograms per gram of body weight given intramuscularly. Phalloidin acts by binding actin, a cytoskeletal protein [Russo et al. Am. J. Pathol. 109:133 (1982)]. Reviews of the chemical structure and toxicology of all toxins derivable from Amanita phalloides including phalloidin have been reported in the literature [Wieland and Wieland, Pharmacol. Rev. 11:87-107 (1959); Wieland, T., Fortschr. Chem. Org. Naturst. 25:214-250 (1967); Faulstich H. and T. Wieland, Eur. J. Biochem. 22:79 (1971); Wieland and Faulstich, Crit. Rev. Biochem. 5:185-260 (1978)].
Sporadically, some investigators have employed phalloidin within in-vitro experiments for its effects upon living cells. For example, the interaction of phalloidin with actin was one of the earliest reported demonstrations [Lengsfeld, A. M., Proc. Natl. Acad. Sci. USA 71:2803-2807 (1974)]. Subsequently, it was shown that phalloidin treatment induces changes in the intercellular junctions of rat hepatocytes [Montsano et al., J. Cell. Biol. 67:310-319 (1975)]. More recently, phalloidin was employed to increase the resistance of Necturus gallbladder epithelium to the passage of an electrical current [Bentzel et al., Amer. J. Physiol. 239:C75-C89 (1980)]. The use of phalloidin in such investigative studies has therefore been primarily as a research tool by which to further characterize and elucidate the mechanism of cytoskeleton action within living cells.
The other major class was identified and founded based on the one naturally occurring substance, antamanide, which was not only completely non-toxic of itself; but also was found capable of actually annulling the toxic effects of fatal doses of phalloidin and/or of protecting the liver completely when administered in therapeutic doses [Wieland et al., Angew. Chem. 80:208 (1968)]. Subsequent investigations of this cyclic decapeptide, antamanide, then proceeded in two different directions: one research effort involved methods of synthesizing, purifying, and preparing analogues of antamanide. These investigations are examplified by: U.S. Pat. Nos. 3,705,887 and 3,793,304; Anderson et al., J. Am. Chem. Soc. 88:1338-1339 (1966); Anderson et al., J. Am. Chem. Soc. 89:5012-5017 (1967); Wieland, T., Angew. Chem. (Internat. Edit.) 7:204-208 (1968); Ovchinnikov et al., Proc. Eur. Pept. Synp. 11:403-415 (1973); Wieland et al., Liebig's Ann. Chem., number 3, pages 371-380, 1977; Burgermeister et al., Eur. J. Biochem. 44:305-310 (1974); Tonelli, A. E., Biochemistry 12:689-692 (1973); Patel, D. J., Biochemistry 12:677-688 (1973); Ivanov et al., Biochem. Biophys. Res. Comm. 42:654-663 (1971); and Bir et al., J. Peptide Protein Res. 13:287-295 (1979)].
In comparison, the other investigations focused upon the physiological and pharmacological attributes of naturally occurring antamanide and its synthetic analogues. These investigations are exemplified by the following: Faulstich et al., Hoppe Scylers. Z. Physiol. Chem. 359:1162-1163 (1974); Ovchennikov et al., Experientia 28:399-401 (1972); Wieland et al., Proc. Nat. Acad. Sci. U.S.A. 81:5232-5236 (1984); Carle, I. L., Proc. Nat. Acad. Sci. U.S.A. 82:7155-7159 (1985); Munter, K. D. and H. Faulstich, Biochim. Biophys. Acta 860:91-98 (1986); Nielsen, O., Acta Pharmacol. Toxicol. 59:249-251 (1986); and Raymond et al., Eur. J. Pharmacol. 138:21-27 (1987).
In all of these published investigations and reports, the cyclic decapeptides comprising antamanide and its analogues were recognized solely as chemical agents capable at very low dosage of counteracting the effects of an absolutely fatal dose of phalloidin or of completely protecting the liver against such a fatal dose of phalloidin. Only recently was there any investigation into areas concerning cell proliferation and wound healing [Choi et al., FASEB J. 3:A290, Abstract No. 368 (1989)]. This Abstract was the first publication to suggest that antamanide might serve as a therapeutic agent in the treatment of vascular disease.
Accordingly, the use of antamanide and the synthetic analogues remains primarily and predominantly as an anti-toxin against the effects of phalloidin; and phallotoxins as a class are known primarily for their toxic effects and, therefore, used primarily as a research tool. There is, thus, no known relationship for these substances with respect to controlling interleukin-mediated edemas in-vivo.